Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis in the United States, and is also designated a Category B food-and waterborne pathogen under the NIAID Biodefense Research Agenda. Case numbers for C. jejuni annually surpass those of Escherichia coli, Shigella and, in some years, even Salmonella species. The Centers for Disease Control have estimated that there are up to 2 million illnesses from campylobacteriosis each year, considering reported and unreported cases. A large number of human cases are acquired by eating contaminated and poorly prepared chicken. Infection of humans results in inflammation of the lower intestinal tract that ultimately involves the colon and rectum. Despite the importance of the organism as a human pathogen, its mechanisms of virulence are poorly understood relative to those of other pathogens. Campylobacter colonization determinants were identified in a signature-tagged mutagenesis screen using a chick infection model. Among the genes identified in this screen were those required for N-linked protein glycosylation system, called pgl genes. The role of protein glycosylation in the biology and pathogenicity-related behavior of C. jejuni is not defined, but systematic genetic analysis of the N-linked glycome of C. jejuni has revealed a subset of these genes that are required for colonization in a chick model. Aim 1 will investigate three of these proteins and determine their roles in both in vivo and in vitro models of C. jejuni pathogenicity as well as the specific effect of protein glycosylation on them. Aim 2 will investigate the chick model further with two specific sub-aims: the first is to analyze the fate of mutant, poorly colonizing bacteria after oro-gastric inoculation and throughout the gastrointestinal tract and the second is to test the hypothesis that mutant bacteria that do not colonize the chicken with wild type efficacy induce a unique innate immune response that contributes to their poor colonization phenotype. Aim 3 will develop and apply assays for screening a library of small molecules for those that inhibit key traits of C. jejuni pathogenicity including protein glycosylation and flagellar assembly. This project will uncover new knowledge about an important human pathogen, Campylobacter jejuni. Experiments are designed to define the role of glycosylated proteins in host-cell association and in two animal models of colonization, including chickens, a natural reservoir of C. jejuni and major source of human infection. The work includes a component that will translate the knowledge from the basic science in this application to development of new reagents for studying and controlling C. jejuni infections.